The present invention relates to nucleic acid sequences which code for sesquiterpene synthases (cyclases) from Grand Fir (Abies grandis), and to vectors containing the sequences, host cells containing the sequences and methods of producing recombinant sesquiterpene synthases and their mutants.
Conifer oleoresin is a mixture of turpentine and rosin that functions in insect defense and in wound sealing (Johnson, M. and Croteau, R. (1987) in Ecology and Metabolism of Plant Lipids (Fuller, G. and Nes, W. D., eds) pp 76-91, ACS Symposium Series 325, American Chemical Society, Washington, D.C.; Gijzen, M., et al., (1993) in Bioactive Volatile Compounds from Plants (Teranishi, R., et al., eds) pp 8-22, ACS Symposium Series 525, American Chemical Society, Washington, D.C.). Turpentine is composed of monoterpene (C10) and sesquiterpene (C15) olefins, while rosin is composed of diterpene (C20) resin acids (FIG. 1). The volatile turpentine fraction of conifer oteoresin, which may consist of up to 30 different monoterpenes (Lewinsohn, E., et al., (1993) Phytochem. Anal. 4, 220-225) and an even larger number of sesquiterpenes (See Example 1, herein) furnishes a solvent for the diterpene resin acids which, upon stem wounding, harden to form a mechanical barrier thereby sealing the wound site (Johnson, M. A. and Croteau, R. (1987) in Ecology and Metabolism of Plant Lipids eds. Fuller, G. and Nes, W. D. (Am. Chem. Soc., Washington, D.C.), ACS Symp. Series 325, pp. 67-91).
Grand fir (Abies grandis) has been developed as a model system for the study of both constitutive and wound-induced oleoresin formation (oleoresinosis). The composition of the monoterpene olefin and the diterpene resin acid fractions of grand fir oleoresin has been defined (Lewinsohn, E., et al., (1993) Phytochem. Anal. 4, 220-225), and the induced biosynthesis of these natural products upon stem wounding has been described in detail (Gijzen, M., et al., (1993) in Bioactive Volatile Compounds from Plants (Teranishi, R., et al., eds) pp 8-22, ACS Symposium Series 525, American Chemical Society, Washington, D.C.; Lewinsohn, E., et al., (1992) in Regulation of Isopentenoid Metabolism (Nes, W. D. , et al., eds) pp 8-17, ACS Symposium Series 497, American Chemical Society, Washington, D.C.; Gijzen, M., et al., (1992) Arch. Biochem. Biophys. 294, 670-674; Funk, C., et al., (1994) Plant Physiol. 106, 999-1005). The time-course of induction, after wounding, of the monoterpene synthases involved in turpentine formation has been analyzed by immunoblotting techniques and the process of induced oleoresinosis was thus shown to involve de novo synthesis of these enzymes (Gijzen, M., et al., (1992) Arch. Biochem. Biophys. 294, 670-674). The cDNA sequence of abietadiene synthase, a diterpene cyclase from grand fir that is involved in resin acid biosynthesis (LaFever, R. E., et al., (1994) Arch. Biochem. Biophys. 313, 139-149)) has been reported (Stofer Vogel, B., et al., (1996) J. Biol. Chem. 271, 23262-23268), and several cDNA clones encoding monoterpene syntheses from this conifer species have recently become available (Bohlmann, J., et al., (1997) J. Biol. Chem. 272, 21784-21792).
In comparison with the monoterpenes and diterpenes of conifer oleoresin, the sesquiterpenes of conifer turpentine have received relatively little experimental attention because they constitute less than 10% of the oleoresin. The relatively low concentrations of sesquiterpenes in conifer oleoresin may, however, belie their biological significance. Sesquiterpenoid phytoalexins, i.e., antibiotic compounds, are well known in angiosperm species (Threlfall, D. R. and Whitehead, I. M. (1991) in Ecological Chemistry and Biochemistry of Plant Terpenoids (Harborne, J B. and Tomas-Barberan, F. A., eds) pp 159-208, Clarendon Press, Oxford, UK), suggesting that the sesquiterpenes of conifer oleoresin may play a similar role in antibiosis.
A conifer oleoresin sesquiterpene that has been relatively well-characterized is juvabione. Juvabione is the methylester of todomatuic acid, an oxygenated derivative of the sesquiterpene olefin bisabolene (FIG. 2). The conifer sesquiterpene juvabione resembles insect juvenile hormones and, thus, can disrupt insect development and reproduction at metamorphosis and diapause (Bowers, W. S., et al., (1976) Science 193, 542-547; Bowers, W. S. (1991) in Herbivores: Their Interaction with Secondary Plant Metabolites, Vol. I, G. A. Rosenthal and M. R. Berenbaum, eds. (Acad. Press, San Diego), pp. 431-456). Juvabione is sometimes referred to as xe2x80x9cpaper factorxe2x80x9d because its presence in paper made from trees of the genus Abies inhibits maturation of insect larvae reared in contact with the paper (Slama, K. and Williams, C. M. (1965) Proc. Natl. Acad. Sci. USA 54, 411-414; Slama, K. and Williams, C. M. (1966) Nature 210, 329-330; Bowers, W. S., et al., (1966) Science 154, 1020-1021). Accumulation of todomatuic acid, the precursor of juvabione, in grand fir after insect feeding suggests that biosynthesis of the juvenile hormone analogue is induced de novo in response to insect attack (Puritch, G. S. and Nijholt, W. W. (1974) Can. J. Bot. 52, 585-587).
Only a single sesquiterpene synthase, E-xcex2-farnesene synthase, from a gymnosperm source, maritime pine (Pinus pinaster), has been reported (Salin, F., et al., (1995) J. Plant Physiol. 146, 203-209). In contrast, several sesquiterpene synthases from angiosperms have been described (Dehal, S. S. and Croteau, R. (1988) Arch. Biochem. Biophys. 261, 346-356; Munck, S. L. and Croteau, R. (1990) Arch. Biochem. Biophys. 282, 58-64; Belingheri, L., et al., (1992) Plant Sci. 84, 129-136), and a number of genes encoding sesquiterpene synthases involved in phytoalexin biosynthesis in angiosperms have been isolated (Facchini, P. J. and Chappell, J. (1992) Proc. Natl. Acad. Sci. USA 89, 11088-11092; Back, K. and Chappell, J. (1995) J. Biol. Chem. 270, 7375-7381; Chen, X.-Y., et al., (1995) Arch. Biochem. Biophys. 324, 255-266).
In accordance with the foregoing, the present invention relates to isolated nucleic acids that encode gymnosperm sesquiterpene synthases, to isolated, recombinant gymnosperm sesquiterpene synthases, to replicable, recombinant expression vectors that include a nucleic acid sequence that encodes a gymnosperm sesquiterpene synthase, to cells that have been transformed, transfected or otherwise manipulated to include one or more nucleic acids of the present invention, and to methods for imparting or enhancing the production of a gymnosperm sesquiterpene synthase in a host cell including the step of introducing into the host cell an expression vector of the present invention under conditions enabling expression of the sesquiterpene synthase protein in the host cell. Representative cDNAs encoding the gymnosperm sesquiterpene synthases E-xcex1-bisabolene synthase, xcex4-selinene synthase and xcex3-humulene synthase have been isolated from Grand Fir (Abies grandis) and sequenced, and the corresponding amino acid sequences have been deduced. Accordingly, in preferred embodiments, the present invention relates to isolated DNA sequences which code for the expression of E-xcex1-bisabolene synthase, such as the sequence designated SEQ ID No:12 which encodes E-xcex1-bisabolene synthase (SEQ ID No:13) from Grand Fir (Abies grandis), for the expression of xcex4-selinene synthase, such as the sequence designated SEQ ID No:19, which encodes xcex4-selinene synthase (SEQ ID No:20) from Grand Fir (Abies grandis), and for the expression of xcex3-humulene synthase, such as the sequence designated SEQ ID No:23, which encodes the xcex3-humulene synthase (SEQ ID No:24) from Grand Fir (Abies grandis).
In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence which codes for a E-xcex1-bisabolene synthase, xcex4-selinene synthase or xcex3-humulene synthase. The present invention is also directed to a base sequence sufficiently complementary to at least a portion of a nucleic acid encoding a E-xcex1-bisabolene synthase, xcex4-selinene synthase or xcex3-humulene synthase, to enable hybridization therewith. The aforesaid complementary base sequences include, but are not limited to: antisense RNA complementary to all or part of a nucleic acid sequence encoding a E-xcex1-bisabolene synthase, xcex4-selinene synthase or xcex3-humulene synthase; fragments of DNA that are complementary to part of a nucleic acid sequence encoding an E-xcex1-bisabolene synthase, xcex4-selinene synthase or xcex3-humulene synthase, and which are therefore useful as polymerase chain reaction primers, or as probes for genes encoding E-xcex1-bisabolene synthase, xcex4-selinene synthase or xcex3-humulene synthase, or related genes.
In yet other aspects of the invention, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention. Thus, the present invention provides for the recombinant expression of gymnosperm E-xcex1-bisabolene synthase, xcex4-selinene synthase and xcex3-humulene synthase, preferably Grand fir (Abies grandis) E-xcex1-bisabolene synthase, xcex4-selinene synthase and xcex3-humulene synthase, and the inventive concepts may be used to facilitate the production, isolation and purification of significant quantities of recombinant E-xcex1-bisabolene synthase, xcex4-selinene synthase and xcex3-humulene synthase (or of their primary enzyme products) for subsequent use, to obtain expression or enhanced expression of E-xcex1-bisabolene synthase, xcex4-selinene synthase or xcex3-humulene synthase in plants, microorganisms or animals, or may be otherwise employed in an environment where the regulation or expression of E-xcex1-bisabolene synthase, xcex4-selinene synthase or xcex3-humulene synthase is desired for the production of these synthases, or their enzyme products, or derivatives thereof.